This invention is directed to the apparatus and techniques used in the preparation of tissue for microscopic analysis using the technique of frozen sectioning. The invention offers rapid and superior quality preparation while saving time, reducing tissue wastage and improving precision to augment the pathologists ability to diagnose the most difficult tissue samples.
In many commonly performed surgical procedures, pathologists or other trained medical professionals are called upon to render rapid interpretation of tissue samples so that the surgeon can make a decision on how to proceed with the operation. The pathologist may be asked to diagnose a disease such as a tumor or an infection or to aid in evaluating the extent of a disease. Commonly, in tumor processes, he is asked to determine involvement of resection margins to answer the question of whether the surgeon has completely removed all of the diseased tissue or still needs to remove more. In order for the pathologist to provide this rapid interpretation, he must use a technique known as frozen sectioning. In many cases, the information needed by the surgeon demands a degree of precision which is difficult or impossible to provide due to the limitations of the available techniques of embedding and freezing.
Frozen sectioning refers to the ultra-thin slicing of tissue to a thickness that is usually about 5-7 mm using an instrument called a cryostat. A cryostat is a refrigerated device, which cools a cutting instrument, known as a cryotome, so that frozen tissues can be cut at the desired thickness. Tissue prepared for microscopic examination by pathologists (and other scientists and technologists) must be hardened to a degree where it can be cut extremely thin. In routine tissue preparation this is accomplished by a time consuming process of fixation, dehydration, solvent infiltration, paraffin infiltration, embedding in blocks of paraffin following which the sample can be cut, stained and interpreted under the microscope. This process takes hours at the minimum and is not suitable for quick, intraoperative interpretation, which needs to be accomplished in a period of minutes, so that the surgeon can make the therapeutic decisions on how to proceed with the surgery.
A faster method of hardening a tissue sample for rapid cutting and preparation of slides is the technique of frozen sectioning. This technique involves hardening the tissue sample by freezing and cutting it on the cryotome, after which a sample can be stained and read. This process can be performed in less than 10 minutes.
The preparation of tissue (freezing and embedding, i.e. cryoembedding) prior to sectioning (cutting on the cryotome) can be performed in several ways. In one method, a cryostat is typically fitted with an area for freezing a chuck (specimen holder). This can be a simple bar or surface on which to sit a flat bottomed chuck or a hole in the surface in which to place the stem of a chuck having a stem. The typical process involves placing a quantity of mounting medium on the chuck and then placing the tissue face up on the medium. The chuck is set upon the cold freezing surface and is cooled from the underside. Often, a flattening cold weight, sometimes referred to as a heat extractor, is placed on top of the specimen to flatten the surface and aid in freezing the sample. The resulting specimen surface is not parallel to the chuck and varies from preparation to preparation. The face of the prepared sample then requires considerable trimming using the cryotome until the desired section is available. This method functions adequately in large samples where tissue wastage is inconsequential and where precision is not critical. However, this method can be difficult or impossible to apply accurately to minute, irregular, flimsy, rubbery and liquefied specimens without undesirable tissue loss or uninterpretable results.
Other methods of preparing tissue include immersing a face up chuck with applied tissue into liquid nitrogen or a dry ice slurry for rapid freezing. Although rapid freezing is possible, precise orientation remains a drawback. Attempts at instrumentation resulting in face-down embedding, a surface flattening process that uses various freezing devices, have improved some aspects of the process yet still do not afford the level of precision necessary to answer all of the surgeons inquiries.
These conventional frozen section preparation techniques are limited in the ability to precisely embed and orient tissue samples. When performed inside the cryostat the process is cumbersome, unpredictable and often results in a block face which is slanted with respect to the tissue face showing portions of the specimen, with other important areas buried in the frozen embedding medium. The block requires significant tissue trimming and wastage in order to approximate the desired section. Often the desired section cannot be achieved because one side has been trimmed away before the more important aspect has been reached. The problem is analogous to trying to uncover an object buried at an angle in the ground. One needs to go much deeper to uncover the deeper side of the object. In the case of a frozen tissue block, considerable attrition occurs when tissue is trimmed away xe2x80x9cdiggingxe2x80x9d for the desired area. Another problem with current methodologies is the difficulty of freezing small or difficult to handle specimens embedded at the proper position in the frozen medium (the process known as embedding) so that the tissue face to be cut will be precisely oriented to yield the desired view on the microscopic slide. As a result, the pathologist is unable to answer the question asked of him.
An additional drawback to current methodologies is that the embedding procedures are often performed while the pathologist is bent over and stretching to do meticulous work inside the deep box-like chamber of the cryostat, a situation not conducive to fine motor tasks or the urgency of the frozen section process. The importance of the questions being asked of the pathologist is of the highest medical importance, as inaccurate results have severe potential for morbidity or mortality. Examples include deciding if a malignant tumor such as a breast or colon cancer has been completely removed where a discrepant result could lead to recurrence, metastasis or death, or deciding if a small skin tumor of the eyelid is completely removed where a discrepant result could lead to recurrence and potential loss of the eye. Such examples are an everyday occurrence in the practice of the pathologist.
The present invention is an apparatus and methodology to provide rapid, precise tissue specimen preparation for use in the frozen section process. This invention prepares multiple specimens at a time with simple preparation, minimal monitoring and results in a highly precise preparation while greatly reducing tissue wastage and affording a level of accuracy that is unprecedented. The apparatus can be applied as a stand-alone instrument, modular units or built into existing or new cryostat designs.
It is an object of the system and method of the present invention to provide a highly precise, rapid preparation of frozen section specimen blocks in a ergonomically comfortable work space so the pathologist can maximize his manual and diagnostic skills to answer any and all questions demanded of him. The features of this apparatus and method accomplish this objective. This apparatus is in its most sophisticated form a cryoembedding station that provides an easy access bench top work area allowing the pathologist complete use of manual skills. The instrument rapidly freezes multiple samples at the same time with a minimum of preparation time and needs no monitoring of the freezing process once the wells are filled. The process of face down embedding on the adherent surface results in flat, predictably and reliably oriented specimen faces that are parallel to the chuck face. This results in greatly reduced trimming and wastage of tissue, and reduces the need for readjustment of the specimen holder angle. The lack of tissue compression by the weighted heat extractor eliminates compression artifacts and slanted surfaces and buried tissue. The blocks that are created are surrounded by an ample xe2x80x9chandlexe2x80x9d of embedding medium resulting in considerable ease in the cutting by frozen sectioning process. The square wells yield blocks with straight sides which reduce tendency to roll when parallel to the blade and provide a easy to manipulate long handle when cut diagonal to the blade. The large wells can yield large flat panoramic sections or can allow for multiple samples. These large samples can be cut with little trimming and wastage due to the flat parallel block faces. The result is that a considerably larger amount of tissue can be prepared with each single preparation, decreasing the number of blocks that need to be cut. This saves time for the pathologist and surgeon and cost for the patient.
This invention also includes and facilitates an original technique, referred to as xe2x80x9cFrozen Block Cryoembeddingxe2x80x9d, which affords a level of precision and accuracy of orientation and embedding previously impossible by conventional methods. The technique allows precise preparation of: (1) minute specimens; (2) thin flat and tubular specimens; (3) torn perforated or friable specimens; (4) flimsy soft specimens difficult to cut with a scalpel; and (5) flattening and orientation of curling, rubbery, angular or curved tissues. The versatility of the apparatus described herein affords a myriad of applications to solve problems in precision embedding and is limited only by the imagination of the pathologists.